The number of receptors and their residence time on the plasma membrane are critical determinants for the response of a cell to extracellular cues and can control cellular plasticity, growth, and differentiation. Cells require endocytosis to retrieve and sort molecules from the plasma membrane as a result of the fusion of regulated and constitutive secretory vesicles. The endocytic pathway can be separated into numerous stages based on the movement of cargo and the identification of morphologically defined compartments. Early events in the endocytic process include membrane invagination and vesicle budding from the plasma membrane. Later events include transport to the late endosome and lysosome for degradation, and recycling back to various compartments. Following transport from early to late endosomes, proteins to be degraded in the lysosome are internalized into the lumen of the late endosome via membrane invagination and vesicle fission. This fission reaction results in the formation of the multivesicular body (MVB) possessing a limiting membrane and intralumenal vesicles. While much progress has been made in elucidating the molecular processes involved in early endocytic events an equally clear understanding of later events remains elusive. Hrs is an endosomal protein that interacts with proteins previously implicated in membrane trafficking. Deletion or mutation of hrs results in an enlarged endosomal phenotype in mouse, fly, and yeast suggesting that hrs may play a role in cargo sorting/endosomal trafficking. The focus of this proposal is to understand the role of hrs in the molecular mechanisms of cargo sorting at the MVB and to determine which of its many protein interactions are relevant for this function. To decipher the function of hrs and its binding partners, we propose an integrated approach using biochemical and functional experiments. First, we will analyze hrs protein interactions. We will then utilize a novel cell-free assay that measures receptor sorting into the lumen of late endosome/MVBs to elucidate requirements for sorting. Finally, we will probe the role of proteins recruited by hrs to the MVB membrane in receptor sorting and ask whether these proteins act independently or in conjunction with each other, and whether they act sequentially. A better appreciation of the mechanism of endocytic protein sorting will help in understanding learning and memory and diseases in which protein transport is disturbed like cancer, lysosomal storage disorders, and Huntingtons Disease.